Sweet Wines Produced by an Innovative Winemaking Procedure: Colour, Active Odorants and Sensory Profile

Sweet Wines Produced by an Innovative Winemaking Procedure: Colour, Active Odorants and Sensory Profile M.J. Ruiz, L. Moyano, L. Zea* Department of Agricultural Chemistry, University of Córdoba, Campus de Rabanales, Edificio Marie Curie, 14014 Córdoba, Spain Submitted for publication: October 2013 Accepted for publication: March 2014 Key words: Chamber-drying grapes, colour, aroma, oak chips, musts, sweet wines The colour, aroma-active compounds and sensory properties of sweet wines from Pedro Ximenez grapes produced by means of an innovative winemaking procedure, based on controlled chamber-drying of grapes, partial fermentation of the must (to 4% or 8% vol ethanol) and subsequent accelerated ageing by contact with oak chips, were studied. Fermentation made the musts less brown and more yellow, whereas ageing made them darker and increased their brown, reddish and yellowish hues. Overall, the musts fermented to 8% vol ethanol exhibited higher odour activity values (OAVs). In addition, the musts aged with oak chips were slightly different from those without chips. Expert tasters gave the highest scores to the musts fermented to 8% (v/v) ethanol with 2 g/l of oak chips added. The winemaking process studied would allow the existing range of sweet wines from dried grapes to be expanded by using a fast, flexible, hygienic procedure. INTRODUCTION In recent years, Pedro Ximenez sweet wine has been under high demand from consumer and the volume produced is virtually sold out every season. The first and foremost step in the production process involves sun-drying the grapes, which face a high risk of deterioration from insect attacks, potential rain and nocturnal dew. In addition, these ambient conditions can favour the production of fungal toxins such as ochratoxin A (OTA), which have an adverse impact on the health and safety of Pedro Ximenez. Unquestionably, increased control over the conditions of grape drying result in better organoleptic and sanitary properties of the final musts. In this respect, Ruiz et al. (2010) have shown that chamber-drying grapes under controlled thermohygrometric conditions provides raisins of substantially improved quality. Chamber-drying for grapes is a fast and reliable method, independent of meteorological conditions. The second important issue to consider is the high levels of sugars found in musts from dried grapes. High sugar content alters the metabolic activity of yeast and can delay, or even stop, alcoholic fermentation. Moreover, fermentation in sugar-rich media is known to lead to wines with high volatile acidity and sometimes with organoleptic faults, as outlined by García-Martínez et al. (2011). Using yeasts that are tolerant of high sugar and ethanol concentrations allows a rapid and reliable fermentation, reducing the risk of sluggish or stuck fermentation and microbial contamination. Logically, the selected yeast strain and the moment of when the fermentation is stopped can affect the wine quality in terms of composition and sensory profile. Furthermore, the OTA contents in wines can decrease during fermentation (Pérez-Serradilla & Luque de Castro, 2008). Another important consideration is the oxidative ageing in oak barrels. During this period of several years, wine acquires its characteristic bouquet as a result of significant changes due to different phenomena: esterification/ hydrolysis and redox reactions, spontaneous clarification, CO 2 elimination, slow and continuous diffusion of oxygen through wood pores, and the extraction of tannins and aromatic substances from the wood to the wine (Camara et al., 2006). The different volatile compounds extracted from wood during this process (lactones, furanic compounds, vanillin derivatives, and phenol derivatives) have important sensory properties and contribute to the overall aroma of the wine. However, ageing in oak casks takes a long time and is a very expensive process. A more economical alternative is the use of oak wood fragments. This practice first appeared in wines produced in emerging countries and later became authorised in the European Union (EU). Several studies have shown the technical possibilities of this practice (Guchu et al., 2006; Rodríguez-Bencomo et al., 2008). In addition, a recent study has shown that wines made with oak wood fragments are scarcely rejected by consumers (Pérez- Magariño et al., 2011). However, the sweetness, chromatic adjustment, aroma profile and complexity of the finally *Corresponding author: qe1zecal@uco.es [Fax: +34957212146] Acknowledgements: The authors are very grateful to Dr Carmen Millán, for her help in selecting and inoculating the yeast used. This work was funded by Spain s Ministry of Science and Technology 205

Innovative Elaboration of Sweet Wines 206 wines depends of the ageing strategy. Therefore, different factors such as doses, size, form and toast level of fragments, ageing time and maceration conditions, must be studied to obtain wines with desirable sensory properties. In this work, the colour, aroma profile and sensory properties of sweet wines from Pedro Ximenez grapes produced by an innovative winemaking procedure, based on chamber-drying of the grapes, partial fermentation of the must by osmo-ethanol-tolerant yeasts, and subsequent accelerated ageing by contact with oak chips, was studied. The results show that is possible to produce high-quality sweet wines from raisins by the fast, cheap and hygienic method proposed. MATERIALS AND METHODS MUST SAMPLES Figure 1 depicts the experimental procedure described below. In this study, 200 kg of ripe Pedro Ximenez grapes were collected in the Montilla-Moriles region (southern Spain). Three batches of grapes of 25 kg each one were distributed uniformly (14 kg/m 2 ) in a single layer and dried in a chamber (Frisol Climatronic, Spain) at air temperatures of 40ºC and humidity of 20%. Samples were collected periodically and the sugar content of the grapes was measured by the Luff-Schoorl method (EEC, 1990). This method is based on the reduction of copper (II) ions in alkaline solution by the reducing sugars, followed by back titration of the remaining copper. The drying was concluded when the sugar concentration was around 450 g/l. The grapes were crushed and subsequently pressed in a vertical press similar to those used at the industrial level (EG-250 Sanahuja, Castellón, Spain). The highest pressure reached in each pressing cycle was 300 bars, and each grape batch was pressed in three cycles in a thermostatised chamber at 20ºC. Inoculation and fermentation A total of 21 L of must was supplied with SO 2 at a concentration of 100 mg/l, blended and distributed among seven 5 000 ml Erlenmeyer flasks containing 3 L of must each. The samples were inoculated with a Saccharomyces cerevisiae strain X5 (CECT13015) previously isolated during spontaneous fermentation of musts from partially dried Pedro Ximenez grapes and chosen on the grounds of its tolerance to high sugar and ethanol concentrations in a previous experiment conducted at the Department of Microbiology of the University of Córdoba (García-Martínez et al., 2011). The starter cultures were prepared by growing each strain separately in YPD medium at 28ºC for 2 h, which was followed by centrifugation and washing with distilled water. Six flasks (two triplicates) were inoculated with 5 x 106 cells/ml and incubated at 25ºC, and the remaining 3 L of must was fortified to 18% (v/v) ethanol and stored at 4ºC in a cold chamber for use as the control. The first three flasks were withdrawn from the chamber FIGURE 1 Experimental design followed in the study.

207 Innovative Elaboration of Sweet Wines when the ethanol content reached 4% (v/v), which occurred after four days of incubation. The other three were withdrawn at an ethanol content of close to 8% (v/v), 14 days after incubation. The fermentation process was monitored by the spectrophotometric method to measure ethanol (Crowell & Ough, 1979). Both batches were immediately fortified to 18% vol with wine alcohol (Alcoholes del Sur, Córdoba, Spain; CE 200-578-6), centrifuged at 3 000 rpm at 4ºC for 10 min and stored in a cold chamber at 4ºC until analysis. Accelerated ageing The accelerated ageing experiment was conducted on 3 000 ml of each fermented must, which was distributed among six 1 000 ml flasks to obtain two batches consisting of six 500 ml samples each. Three flasks in each batch were supplied with American oak in the form of medium-toasted chips (Anatride Ibérica SL, Zaragoza, Spain) at a concentration of 1 g/l, and the other three with a 2 g/l concentration of identical chips. The flasks were then stoppered with hydrophobic cotton and allowed to stand in a thermostated room at 20 C for 30 days, with shaking by hand on a daily basis. After the experiment was finished, the wood chips were removed and the samples were stored in a cold chamber at 4 C until analysis. Conventional analyses The ph, total and volatile acidities and reducing sugars were determined according to EEC (1990) methods. Glycerine was quantified by direct injection of samples in a gas chromatograph HP 6890 GC System (Agilent Technologies, CA, USA) equipped with a capillary column with molten silica CP-WAX 57 CB (50 m x 0.25 mm x 0.4 μm thickness), and a FID was used as the detector according to Peinado et al. (2004). Browning and colour evaluation Browning of the samples was measured as absorbance at 420 nm. Colour was determined according to the recommendations of the International Commission on Illumination (CIE, 2004) with the illuminant D65 (daylight source) and 10º standard observer (perception of a human observer). The parameters calculated were a* (red/green values), b* (yellow/blue values), and L* (lightness). From the CIELab space, other psychophysical parameters were calculated, such as C* (chroma or saturation) and h (hue angle). All the respective measurements were carried out in triplicate in a Perkin-Elmer Lambda 25 model spectrophotometer (USA) using 10 mm quartz tray after filtering the samples through a HA-0.45 μm paper (Millipore). Identification and quantification of aroma compounds Each aroma compound was identified by means of its retention time, co-eluted with a standard solution of commercial product (Sigma Aldrich, Munich, Germany), and confirmed by mass spectrometry (Hewlett-Packard 5972 MSD, Agilent Technologies, CA, USA). Positive ion electron impact mass spectra were acquired in scan mode, with a range of m z 39 to 300, and a scan rate of 1.6 scans/sec. For each compound the mass spectra were confirmed by comparison with the Wiley mass spectral library. The chromatographic column, injector and oven temperatures, carrier gas and its flow were the same as those used for the quantification, as described below. For the quantification of the aroma compounds, samples of 100 ml were adjusted to ph 3.5, 150 mg of 2-octanol was added as an internal standard and then extracted with 100 ml of freon-11 (Sigma-Aldrich Química S.A., Madrid, Spain) in a continuous extractor for 24 hours. After concentration of the freon extracts to 0.2 ml in a Kuderna-Danish microconcentrator, 3 μl were injected into the Hewlett-Packard 5890 series II chromatograph with an HP-INNOWax column of 60 m x 0.32 mm x 0.25 μm thickness (Agilent Technologies, CA, USA), equipped with a split/splitless injector and an FID detector. The oven temperature programme was as follows: 5 min at 45ºC, 1ºC/min up to 185ºC and 30 min at 185ºC. Injector and detector temperatures were 275ºC and 300ºC respectively. The carrier gas was helium at 70 kpa and was split 1:30. The quantification was made by using chromatographic response factors, calculated for each compound in relation to the internal standard, in standard solutions of commercial products (purity > 95%) supplied by Sigma Aldrich (Munich, Germany). The quantification was done in triplicate. Odour descriptors of aroma compounds For the determination of odour descriptors, a direct olfaction of the pure reference standards (Sigma Aldrich, Munich, Germany) was conducted on water solutions of each compound with a concentration slightly higher than its perception threshold (10%). The taste panel consisted of 20 trained judges of both sexes (12 female and 8 male) between the ages of 20 and 55 years (ISO 5496:1992) from the University of Cordoba. All judges were trained in preliminary sessions using reference standards taken from Sigma-Aldrich (Munich, Germany) and from Le nez du vin (Jean Lenoir, Provence, France) according to ISO 5496:1992. Thirteen judges of the above-mentioned panel had previous experience in the sensory evaluation of sherry wine. During the training, five standards were tasted per session and were discussed by the judges in terms of odour descriptors, and consensus on the terms was reached by eliminating those that were considered irrelevant or redundant. Later, five different odour samples were served at each session and there were 13 evaluation sessions. Samples were prepared 30 min before the test to allow time for the vapour pressure to reach equilibrium at ambient temperature. The odour substances (1 ml) were poured directly into the glass flasks containing a piece of cotton and were closed immediately. Evaluation was conducted in our laboratory in individual booths at room temperature (25ºC). The responses of the judges were compiled for all aroma compounds, and those odour descriptors cited by less than 15% of the panel were eliminated. The odour descriptors are listed in Table 1. Sensory analysis of wines The partially fermented musts treated with oak wood chips were subjected to a quantitative descriptive analysis (QDA) to establish their sensory profile (Stone et al., 2012). The tasting panel was composed of five experts (three men and two women, 40 to 50 years old), selected by the Quality Regulation Board of the Montilla-Moriles designation

Innovative Elaboration of Sweet Wines 208 TABLE 1 Odour descriptors, odorant terms (Wine Aroma Wheel) and perception thresholds (µg/l) of the aroma compounds determined in the sweet wines. Compound Odour descriptor Odorant terms Threshold Ethyl acetate Pineapple, varnish, anise Tropical fruit, pungent, spicy 7 500 a 1,1-Diethoxyethane Green fruit, liquorice Tree fruit, spicy 1 000 a Propyl acetate Glue, Christmas sweet Chemical, caramelised 65 000 a 2,3-Butanedione Buttery Caramelised 100 a Ethyl propanoate Apple Tree fruit 5 000 a Isobutyl acetate Sweet, apple, banana Caramelised, tree fruit, tropical fruit 6 140 a 2-Butanol Vinous Chemical 1 000 000 a 2,3-Pentanedione Buttery Caramelised 1 000 b Butyl acetate Ripe pear, glue Tree fruit, chemical 4 600 a Hexanal Green Fresh 350 c Isobutanol Alcohol, nail polish Chemical, pungent 40 000 a Isoamyl acetate Banana Tropical fruit 30 a 1-Butanol Medicinal Phenolic 820 000 a Isoamyl alcohols Alcohol, nail polish Chemical, pungent 65 000 a Ethyl hexanoate Banana, green apple Tropical fruit, tree fruit 5 a Isoamyl butanoate Banana Tropical fruit 1 000 c Hexyl acetate Apple, banana Tree fruit, tropical fruit 1 000 b Octanal Soapy, fatty, honey, grass Chemical, oily, caramelised, fresh 640 a Acetoin Buttery, cream Caramelised 30 000 a Ethyl heptanoate Sweet, strawberry, banana Caramelised, berry, tropical fruit 10 000 d 3-Methylpentanol Pungent, vinous, cacao, herbaceous Pungent, chemical, caramelised, fresh 50 000 a Ethyl lactate Strawberry, raspberry, buttery Berry, caramelised 100 000 a 1-Hexanol Green, grass Fresh 8 000 a E-3-hexenol Grass, resinous, cream Fresh, resinous, caramelised 1 000 c 3-Etoxypropanol Overripe pear Tree fruit 50 000 a E-2-hexenol Green Fresh 15 000 c Furfural Burned almond, floral Burned, floral, marshmallow 15 000 a Ethyl 3-hidroxybutanoate Fresh, grape Fresh, berry, floral 67 000 a Benzaldehyde Bitter almond, nutty, smoky Nutty, burned 5 000 a Isobutanoic acid Rancid butter Lactic 20 000 a 5-Methylfurfural Bitter almond, spicy Nutty, spicy 16 000 a γ-butyrolactone Coconut, caramel Tropical fruit, caramelized 100000 a Butanoic acid Rancid, cheese Lactic 10000 a Furfuryl alcohol Medicinal Phenolic 15000 a Diethyl succinate Lavender, overripe melon Caramelized, floral, tropical fruit 100000 a 3-Methylbutanoic acid Parmesan cheese, rancid Lactic 3000 a α-terpineol Lilac Floral 38000 a γ-hexalactone Coconut, almond liqueur, sweet Tropical fruit, nutty, caramelised 359 000 d Methionol Cut hay, cooked potato Fresh 500 a Geranial Citrus, sweet Citrus, caramelised 1 000 c Nerol Herbaceous, lemon balm Fresh, floral 10 000 c γ-heptalactone Coconut, herbaceous, caramel Tropical fruit, fresh, caramelised 1 000 c 2-Phenylethanol acetate Rose, honey Floral, caramelised 250 a Hexanoic acid Cheese Lactic 3 000 a Guaiacol Smoky Burned 20 e Benzyl alcohol Fruity, walnut Tree fruit, nutty 900 000 a E-oak lactone Coconut, burned wood, vanilla Tropical fruit, burned, spicy 122 a

209 Innovative Elaboration of Sweet Wines TABLE 1 (CONTINUED) Compound Odour descriptor Odorant terms Threshold 2-Phenylethanol Rose, honey Floral, caramelised 10 000 a Z-oak lactone Coconut, burned wood, vanilla Tropical fruit, burned, spicy 35 a Methyleugenol Clove Spicy 10 000 d 4-Ethylguaiacol Smoky, clove Burned, spicy 46 a Diethyl malate Peach, prune Tree fruit 760 000 a Pantolactone Liquorice, smoky Spicy, burned 500 000 a Octanoic acid Oily, rancid Oily, lactic 8 800 a 2-Phenylethanol hexanoate Overripe banana, sweet Tropical fruit, caramelised 50 000 c Eugenol Clove Spicy 5 a γ-decalactone Peach Tree fruit 1 000 a 4-Ethylphenol Spicy Spicy 140 000 a Syringol Smoky Burned 1 700 e Decanoic acid Rancid, waxy Lactic, oily 15 000 a Farnesol Fruity, balsamic, floral, clove Tree fruit, fresh, floral, spicy 72 000 c Isoeugenol Clove, burned wood, sweet Spicy, burned, caramelised 6 c Monoethyl succinate Burned caramel, coffee Burned 1 000 000 c Vanillin Vanilla Spicy 65 e 2,3-Butanediol Sweet, creamy, butter Caramelised 668 000 a a = from Zea et al. (2007) b = from Chaves et al. (2007) c = determined by the author in alcoholic solution; data not published. Five solutions of ascending concentration of these compounds were used. Starting from the lowest concentration solution, the judges indicated odorant sensations different to that perceived in the control (distilled water), according to the ISO 5495:1983. d = from Moreno (2005) e = from Moyano et al. (2012) of origin (southern Spain). These judges are professional oenologists who know the characteristics of typical wines produced in the region very well, and therefore are sensitive to even small differences. The tasting session was preceded by another one to reach consensus on the sensory attributes used in the sensory analysis of the sweet musts studied (fermentative aroma, woody, astringency, sweetness, acidity and colour). The performance of the expert tasters was accepted as highly reliable and consistent according to the abovementioned Quality Regulation Board. In three consecutive sessions, the tasters examined the seven samples of wine three times, with the ones being presented in random order each time. Their sensory attributes, sensory balance and global impression were scored using the scale method in accordance with ISO 4121:1987. The scale direction goes from left to right with increasing intensities: 1 (imperceptible), 2 (weak), 3 (moderate), 4 (strong) and 5 (excessive) (Stone et al., 2012). Balance and global impression were measured on a five-point hedonic scale, from 1 (unacceptable) to 5 (highly acceptable), with three intermediate points. The results were given as mean ± standard deviation. The means of the sample scores were shown in a spider web graph. Samples were stored in a refrigerator and withdrawn one hour before the sensory test in order to facilitate the adjustment in temperature to that of the tasting room. Evaluation was carried out in a thermostated room with individual booths at 20 C. Twenty millilitres of sample were used in standardised wine glasses (ISO 3591:1992). These were marked with a code and covered to avoid any loss of organoleptic properties. The sequence of sample presentation went from wines with 4% (v/v) to those with 8% (v/v) alcohol content. All the samples were evaluated in a single session, one at a time, with a wait of 3 min between samples. Statistical treatments ANOVA (LSD 95% level) was performed on the triplicates of winemaking variables studied. The OAVs for odorant term and the results of the sensory analysis of the wines were calculated as mean ± standard deviation of three samples for each wine type. To find which variables contributed most to the difference in the aroma profiles of the wines, a PCA was performed on the triplicates of OAVs of odorant terms. The values of the variables were standardised by subtracting their means and dividing by their standard deviations. Results were validated by full internal cross-validation. Software Statgraphics TM (Version 5.0). STSC Inc, Rockville, MD, USA. RESULTS AND DISCUSSION Winemaking variables Table 2 shows the conventional oenological and colour parameters of the musts. The ANOVA shows that wines with different alcohol levels obtained by fermentation also

Innovative Elaboration of Sweet Wines 210 TABLE 2 Means and standard deviations (n = 3) for conventional oenological and colour parameters of the control must (unfermented), and the musts partially fermented to 4% (v/v) or 8% (v/v) of ethanol and to which oak chips subsequently were added (1 g/l or 2 g/l) for 30 days. Partially fermented Partially fermented + oak chips Control 4% (v/v) 8% (v/v) 4% (v/v) 8% (v/v) 1 g/l 2 g/l 1 g/l 2 g/l ph # 3.93 ± 0.01 3.64 ± 0.01 3.61 ± 0.04 3.53 ± 0.01 3.53 ± 0.02 3.72 ± 0.01 3.74 ± 0.01 Total acidity (meq/l) # 51.0 ± 0.3 67.8 ± 0.3 86.9 ± 0.1 71 ± 1 71 ± 1 95 ± 2 94.5 ± 0.8 Volatile acidity (meq/l) # 7.9 ± 0.3 18.0 ± 0.3 39.3 ± 0.5 19.8 ± 0.1 19.8 ± 0.1 39.5 ± 0.7 39.5 ± 0.7 Reducing sugars (g/l) # 450 ± 2 345 ± 5 301 ± 4 349 ± 1 348 ± 3 302 ± 4 301 ± 4 Glycerine (g/l) # 5.2 ± 0.4 18.5 ± 0.9 25 ± 1 19 ± 1 18.7 ± 0.9 25 ± 1 25 ± 1 Absorbance at 420 nm 0.640 ± 0.002 0.560 ± 0.003 0.555 ± 0.003 1.018 ± 0.001 1.065 ± 0.004 1.034 ± 0.006 1.084 ± 0.002 a* 1.62 ± 0.09 0.07 ± 0.02-0.16 ± 0.02 6.8 ± 0.2 6.76 ± 0.01 7.2 ± 0.1 7.30 ± 0.03 b* 38.14 ± 0.04 33.6 ± 0.7 33.6 ± 0.2 52.22 ± 0.05 53.41 ± 0.01 50.56 ± 0.06 51.72 ± 0.04 L* 89.63 ± 0.06 91.07 ± 0.06 91.53 ± 0.06 81.30 ± 0.01 81.10 ± 0.01 80.63 ± 0.06 80.40 ± 0.01 C* ab 38.17 ± 0.03 33.6 ± 0.7 33.6 ± 0.2 52.66 ± 0.04 53.84 ± 0.01 51.08 ± 0.08 52.23 ± 0.04 h ab 87.6 ± 0.1 89.8 ± 0.1 90.21 ± 0.09 82.6 ± 0.2 82.78 ± 0.01 81.8 ± 0.1 81.97 ± 0.04 # Values different for the wines fermented to 4% and 8% (v/v) ethanol at 95% ANOVA level presented values of ph, total and volatile acidity, reducing sugar and glycerine that were different in each case at the LSD 95% level. This shows that the fermentations progressed successfully. As can be seen in Table 2, alcoholic fermentation lowered the ph, and increased the total and volatile acidity markedly. This suggests that, as previously found by other authors (Pigeau et al., 2002; Erasmus et al., 2004; Malacrino et al., 2005), the yeasts produced increased amounts of acetic acid in response to the osmotic stress caused by high sugar levels. In general, high levels of volatile acidity are not considered positive for quality of wine. However, for the sweet wines, the increased production of acetic acid countered the overall sweetness in the end-product through its contribution to total acidity (Pigeau et al., 2007). Obviously, the levels of reducing sugars decreased as a result of fermentation, especially in the musts fermented to 8% (v/v) ethanol (about 300 g/l) and irrespective of the ageing procedure. Also, glycerine increased markedly, with average concentrations close to 18 and 25 g/l in the musts fermented to 4 and 8% respectively, even after ageing. These high glycerine levels are the result of the osmotic stress of the yeasts. Glycerine has been proposed by several authors to be one of the most compatible metabolites to equilibrate the osmotic pressure in the cell with the use of glycerine-3- phosphate dehydrogenase. Fermentation decreased the colour-related parameters, A 420, a *, b * and C ab*, and increased L * and h ab ; the resulting sweet wines were thus less brown and more pale. This may have been the result of yeasts adsorbing some coloured compounds during fermentation (Mérida et al., 2005) and/or of low β-glucosidase activity in the yeasts. On the other hand, accelerated ageing increased A 420, a *, b * and C ab*, and decreased L * and h ab, with the resulting wines exhibiting increased brown, dark, reddish and yellowish hues, especially at the higher wood chip concentration. This dissimilar behaviour may have been the result of oxidation, condensation and/or polymerisation reactions, and also of the extraction of mainly phenolic compounds from the chips. Aroma compounds Table 3 lists the contents of the compounds studied in the sweet wines. The odour activity value (OAV) for each compound was calculated by dividing its concentration in the samples by the concentration corresponding to its odour threshold (Table 1). Over the past few years, numerous authors have proposed an approximation of the importance of a flavour compound in the wine based on the OAV. However, quantifying the perceived intensity of odorants and their contribution to the overall aroma is more complex. Based on the perception thresholds shown in Table 1, there were only 12 active odorants (OAV > 1) in at least one sample. 2,3-Butanedione (diacetyl) is one of the typical odorants in musts from dried Pedro Ximenez grapes (Ruiz et al., 2010), which it endows with buttery notes. The musts fermented to 4% (v/v) ethanol exhibited higher OAVs (approximately 29.5), which suggests that the point at which the fermentation process is stopped influences the final concentrations of this compound. It is most likely that the reductive conditions prevailing at the end of alcoholic fermentation facilitate the reduction of 2,3-butanedione to 2,3-butane-

211 Innovative Elaboration of Sweet Wines TABLE 3 Means and standard deviations (n = 3) for the aroma compounds (µg/l) detected in the control must (unfermented), and the musts partially fermented to 4% (v/v) or 8% (v/v) of ethanol to which oak chips subsequently were added (1 g/l or 2 g/l) for 30 days. Partially fermented Partially fermented + oak chips Compound Control 4% (v/v) 8% (v/v) 4% (v/v) 8% (v/v) 1 g/l 2 g/l 1 g/l 2 g/l Ethyl acetate 24 095 ± 572 51 213 ± 8 870 110 324 ± 10 015 38 983 ± 1 277 41 863 ± 2 340 106 666 ± 7 637 113 333 ± 5 773 1,1-Diethoxyethane 1 274 ± 26 2 123 ± 178 4 595 ± 415 1 624 ± 53 1 744 ± 98 4 610 ± 212 4 795 ± 193 Propyl acetate 8.0 ± 0.7 8 ± 1 12 ± 2 11 ± 3 15 ± 4 16 ± 2 14 ± 1 2,3-Butanedione 1 215 ± 153 2 952 ± 100 972 ± 50 4 873 ± 221 5 068 ± 540 1 531 ± 112 1 656 ± 230 Ethyl propanoate 20 ± 3 30 ± 6 45 ± 5 28 ± 2 24 ± 5 40 ± 2 43 ± 6 Isobutyl acetate 40 ± 5 65 ± 9 94 ± 18 nd nd nd nd 2-Butanol 9 ± 2 26 ± 3 7.4 ± 0.6 nd nd nd nd 2,3-Pentanedione 20 ± 5 398 ± 28 47 ± 1 405 ± 30 435 ± 52 46 ± 4 47 ± 8 Butyl acetate nd 92 ± 14 128 ± 4 154 ± 4 127 ± 9 122 ± 5 134 ± 6 Hexanal 41 ± 7 29 ± 2 6.0 ± 0.7 45 ± 5 35 ± 4 9 ± 1 23 ± 4 Isobutanol 11 396 ± 2 582 30 483 ± 2 659 40 896 ± 2 517 25 170 ± 3 110 28 293 ± 3 827 36 210 ± 3 200 38 330 ± 1 703 Isoamyl acetate 52 ± 6 158 ± 10 208 ± 13 183 ± 10 191 ± 10 286 ± 18 295 ± 26 1-Butanol 461 ± 42 1 067 ± 52 1 509 ± 74 1 768 ± 60 1 121 ± 41 1 110 ± 79 1 246 ± 256 Isoamyl alcohols 18 204 ± 3 273 102 473 ± 7 322 125 593 ± 6 630 89 283 ± 6 315 97 340 ± 3 440 146 721 ± 2 944 132 378 ± 3 951 Ethyl hexanoate nd 86 ± 8 128 ± 9 101 ± 10 101 ± 19 144 ± 13 147 ± 12 Isoamyl butanoate 87 ± 5 89 ± 4 80 ± 13 82 ± 9 72 ± 4 40 ± 6 44 ± 5 Hexyl acetate 32 ± 5 15 ± 1 35 ± 2 23 ± 2 25 ± 3 14 ± 4 16 ± 2 Octanal 53 ± 4 72 ± 16 49 ± 6 127 ± 5 91 ± 14 71 ± 8 46 ± 2 Acetoin 247 879 ± 18 859 1 228 524 ± 216 045 151 285 ± 6 803 1 415 982 ± 96 303 1 422 045 ± 86 768 253 803 ± 21 226 229 211 ± 16 552 Ethyl heptanoate 46±5 40±5 44±2 29±7 18 ± 2 28 ± 9 16 ± 3 3-Methylpentanol nd 27±3 24±5 24±7 22±5 28±2 30±2 Ethyl lactate 1225±131 1758±129 4073±460 5239±351 5016±226 14714±1114 14041±1519 1-Hexanol 18±3 28±7 28±6 32±6 31±6 45±6 40±6 E-3-hexenol 5.8 ± 0.7 nd 16 ± 2 nd nd 9 ± 1 7.6 ± 0.9 3-Ethoxypropanol 9 827 ± 471 11 665 ± 414 12 793 ± 1 951 12 349 ± 194 11 091 ± 544 17 374 ± 2 005 14 382 ± 1 713 E-2-hexenol 7 ± 1 24 ± 2 16 ± 1 33 ± 4 44 ± 5 32 ± 3 22 ± 2 Furfural 40 ± 1 65 ± 7 110 ± 20 1 150 ± 200 1 840 ± 230 1 035 ± 210 1 576 ± 223 Ethyl 3-hidroxybutanoate 46 ± 8 48 ± 10 62 ± 4 27 ± 4 21 ± 3 45 ± 8 48 ± 8 Benzaldehyde 24 ± 2 50 ± 10 17 ± 1 26 ± 2 22 ± 2 18 ± 2 13 ± 4 Isobutanoic acid 820 ± 100 12 555 ± 953 19 593 ± 616 11 019 ± 780 11 834 ± 1 470 18 263 ± 2 344 15 712 ± 2 003 5-Methylfurfural 35 ± 4 45 ± 5 43 ± 5 1 020 ± 83 2 130 ± 106 1 062 ± 175 2 400 ± 360 γ-butyrolactone 2 436 ± 229 2 596 ± 343 5 821 ± 818 3 808 ± 255 4 381 ± 114 10 185 ± 712 12 200 ± 704 Butanoic acid 97 ± 14 260 ± 15 152 ± 36 137 ± 24 85 ± 14 86 ± 6 70 ± 5

Innovative Elaboration of Sweet Wines 212 TABLE 3 (CONTINUED) Partially fermented Partially fermented + oak chips Compound Control 4% (v/v) 8% (v/v) 4% (v/v) 8% (v/v) 1 g/l 2 g/l 1 g/l 2 g/l Furfuryl alcohol 4.7 ± 0.7 nd nd nd nd nd nd Diethyl succinate 183 ± 19 198 ± 25 501 ± 21 289 ± 17 268 ± 24 809 ± 94 733 ± 54 3-Methylbutanoic acid 130 ± 11 2 207 ± 92 2 495 ± 150 1 968 ± 162 1 255 ± 121 2 208 ± 346 1 557 ± 74 α-terpineol 12 ± 1 15 ± 2 14 ± 2 9.1 ± 1 10 ± 1 15 ± 1 16 ± 3 γ-hexalactone 19 ± 4 8 ± 1 2.7 ± 0.7 8.3 ± 0.6 7 ± 1 8 ± 1 10 ± 2 Methionol 67 ± 2 65 ± 10 70 ± 9 nd nd nd nd Geranial 56 ± 7 34 ± 6 20 ± 1 13.0 ± 0.2 14 ± 2 20 ± 4 15 ± 1 Nerol 13 ± 2 nd nd nd nd nd nd γ-heptalactone 66 ± 7 67 ± 12 86 ± 27 36 ± 3 43 ± 7 108 ± 26 120 ± 28 2-Phenylethanol acetate 20±2 21±3 29±3 34±4 28±3 45±5 32±7 Hexanoic acid 15±2 64±4 82±7 75±7 68±8 70±20 72±8 Guaiacol 0 0 0 6±1 9±2 5.3±0.6 9.4±0.8 Benzyl alcohol 86±10 152±18 159±23 173±17 143±28 219±11 188±15 E-oak lactone nd nd nd 3.7±0.7 8±1 4.4±0.5 9±2 2-phenylethanol 11 458 ± 1 323 31 036 ± 4 703 69 932 ± 3 229 35 574 ± 4 911 26 260 ± 2 789 78 891 ± 5 066 75 253 ± 4 949 Z-oak lactone nd nd nd 11 ± 1 19 ± 2 13 ± 1 28 ± 6 Methyleugenol nd nd nd 50 ± 8 68 ± 5 44 ± 4 53 ± 4 4-Ethylguaiacol nd nd nd 11 ± 1 19 ± 3 20 ± 2 33 ± 2 Diethyl malate 61 ± 6 129 ± 14 248 ± 23 168 ± 4 183 ± 8 531 ± 51 324 ± 17 Pantolactone 7 ± 1 18 ± 2 20 ± 2 20.8 ± 0.7 17 ± 3 65 ± 4 61 ± 12 Octanoic acid 2.9 ± 0.7 84 ± 8 99 ± 14 62 ± 11 49 ± 5 200 ± 16 183 ± 9 2-Phenylethanol hexanoate 7 ± 1 15 ± 2 11 ± 1 14 ± 1 15 ± 1 18 ± 1 10.9 ± 0.8 Eugenol nd nd nd 16 ± 3 24 ± 3 15 ± 1 23 ± 3 γ-decalactone 20 ± 2 25 ± 3 27 ± 5 32 ± 4 36 ± 2 39 ± 3 33 ± 2 4-Ethylphenol nd nd nd 11 ± 1 10 ± 1 30 ± 2 29 ± 2 Syringol nd nd nd 58 ± 6 69 ± 3 43 ± 3 56 ± 7 Decanoic acid nd 107 ± 8 117 ± 14 140 ± 10 160 ± 15 175 ± 15 185 ± 20 Farnesol nd 7.5 ± 0.5 8 ± 2 18 ± 2 13 ± 2 19 ± 3 21.2 ± 0.7 Isoeugenol nd nd nd 1.9 ± 0.3 2.4 ± 0.4 4.4 ± 0.6 4.3 ± 0.9 Monoethyl succinate nd 51 ± 7 36 ± 6 70 ± 12 53 ± 7 47 ± 1 29 ± 2 Vanillin nd nd nd 51 ± 10 180 ± 30 48 ± 5 185 ± 20 2,3-Butanediol a 0.25 ± 0.03 1.49 ± 0.08 5.03 ± 0.11 1.68 ± 0.08 1.75 ± 0.05 5.05 ± 0.18 4.9 ± 0.7 nd = not detected a = the concentration is expressed as g/l

213 Innovative Elaboration of Sweet Wines diol, thereby diminishing its impact on the aroma (Martineau et al., 1995). The odorant activity of 2,3-butanediol increased markedly during fermentation and the compound reached its highest OAVs ( 7.5) in the must fermented to 8% (v/v) ethanol. This compound is associated with sweet, creamy, buttery notes. The higher alcohols isobutanol, isoamyl and 2-phenylethanol only reached their perception thresholds at the end of fermentation; however, they exhibited near-unity OAVs, so it is reasonable to assume that they can hardly have contributed to the overall aroma of the sweet wines studied. Ethyl hexanoate and isoamyl and ethyl acetates exhibited the highest odorant activity in the partially fermented musts and thus were major contributors to their aroma profile, which they enriched with fruity, anise and varnish notes. Ruiz et al. (2010) previously found ethyl acetate to increase during the drying of Pedro Ximenez grapes, both in the sun and in chambers, through the effect of its involvement in anaerobic metabolism in the grape berries during drying. Also, this compound has been deemed a useful marker for metabolism in drying grape berries (Chkaiban et al., 2007). 1,1-Diethoxyethane (diacetal) slightly surpassed its perception threshold in the unfermented must, and its concentration increased, also slightly, from the effect of fermentation. The most important compound in odorant terms was acetoin (3-hydroxy-2-butanone), with OAVs of about 40 in the musts fermented to 4% (v/v) ethanol. Like 2,3-butanedione, acetoin is a typical component of musts from Pedro Ximenez cv. grapes dried in the sun or in a chamber, and increases during drying. This compound behaved similarly to 2,3-butanedione, but differed even more markedly between the two types of fermented must. Although acetoin is a typical product of alcoholic fermentation, it can also come from other sources, including yeasts and bacteria, or malolactic bacteria. However, sweet dessert wines fortified halfway through fermentation were found to contain more acetoin than identical wines allowed to ferment completely. This has been ascribed to the high levels of acetoin present in the middle of the process, which subsequently decreases from the effect of its conversion to 2,3-butanediol (Herraiz, 1990). 1-Butanol, ethyl lactate, 3-ethoxypropanol, isobutanoic acid, γ-butyrolactone and 3-methylbutanoic acid were present at high concentrations as a result of the alcoholic fermentation, but exhibited no odorant activity in most of the wines studied. None of the other compounds studied reached its perception threshold. As can be seen from Table 3, the compounds exhibiting odorant activity at the end of partial fermentation of the musts remained active during the 30 days of accelerated aging. This was particularly so with 2,3-butanedione and isoamyl acetate, which were the most interesting compounds in terms of OAVs at this stage, irrespective of the wood chip concentration used; both compounds increased in the two types of fermented must. 2,3-Butanedione also increased during the oxidative ageing of Pedro Ximenez sweet musts (Chaves et al., 2007), probably as a result of the oxidation of acetoin or the gradual decrease in the levels of SO 2 because of its addition to the carbonyl groups in diacetyl. The reversible and exothermal nature of this reaction could change the buttery flavour of wines (Bartowsky & Henschke, 2004), an effect that can also be observed in sweet wines. The increase in isoamyl acetate may be related to the high values of acetic acid in the wines in the presence of oak chips, measured as volatile acidity (Table 2). Among the compounds not present in the fermented musts but extracted from the wood chips, only eugenol and vanillin surpassed their perception threshold, and only in a few samples. In this sense, vanillin was only active in the samples treated with a 2 g/l concentration of oak chips, with OAV 3 irrespective of the alcohol content reached by the wines. This compound is one of the most phenolic aldehydes in wine and is responsible for the typical vanilla notes of wines aged in wood casks (Singleton, 1995). Most phenol aldehydes come from the wood and are present at negligible concentrations in the base wine. Therefore, drying and toasting the wood used to age wine influences the extent to which these aldehydes (and, especially, vanillin) are extracted from it. Eugenol was active in the sweet wines treated with a 1 or 2 g/l concentration of oak chips, which imparted a typical clove aroma. Although furfural and 5-methylfurfural form during the grape-drying process (Ruiz et al., 2010), oxidation and the presence of wood chips in the medium increased their contents markedly, albeit below their perception thresholds. However, these compounds might be useful as markers for the ageing process, since their levels are highly correlated with the ageing time (Camara et al., 2006). The (E) and (Z) isomers or oak lactone (β-methyl-γ-octalactone) were only detected in the samples treated with oak chips; however, both exhibited OAV < 1. Other volatile phenols, including guaiacol, 4-ethylguaiacol, 4-ethylphenol, syringol and isoeugenol, exhibited increased contents after the accelerated ageing of the wines. Odorant terms To compare the aroma profiles of the wines studied by considering a small number of variables, we used the OAV for the compound grouped into nine odorant terms according to their similar odour descriptors (caramelised, tropical fruit, tree fruit, spicy, pungent floral, chemical, toasted, and lactic). The remaining terms listed in Table 1 are not significant. The addition of the OAVs of the compounds to each term cannot be interpreted as an arithmetical addition of odorant sensations. Several authors have used odorant terms and aromatic series to establish aroma profiles for musts and wines from different winemaking processes (Zea et al., 2007; Ruiz et al., 2010; Gómez García-Carpintero et al., 2012; López de Lerma et al., 2012). In any case, the proposed method is valid for comparing wines of the same type (sweet wines in this work), because the odorant terms always comprise the same compounds. However, this method of studying the aroma profile has the advantage that it strongly reduces the number of variables to be interpreted, preserving their relative importance according to the OAVs of the compounds assembled. Figure 2 shows the aroma fingerprint of the samples as obtained from the components with OAV > 1. As can be seen, the profile was altered considerably by the fermentation process. Overall, the musts fermented to 8% (v/v) ethanol exhibited higher OAVs; and the caramelised term had a

Innovative Elaboration of Sweet Wines 214 significantly higher OAV in the musts fermented to 4% (v/v) ethanol. After the addition of oak chips, the terms caramelised and also, to a lesser extent, spicy, exhibited an increase in the OAVs in both types of wines, as did the tropical fruit term in those wines with the higher alcohol content. Also, ageing introduced the term toasted, which was absent from the samples to which no wood chips had been added. A principal component analysis (PCA) on the OAVs of the odorant terms considered was conducted in order to identify those with the greatest influence on the aroma profile of sweet wines (Fig. 3). The first two principle components (PCs) jointly accounted for 87% of the overall variance. PC1 explained 72% of the variance and encompassed the tropical fruit, tree fruit, pungent and floral terms, which afforded discrimination according to the alcohol content reached by the samples. The musts fermented to 8% (v/v) ethanol aged with oak chips were slightly different from those without chips. Since these wines exhibited the highest scores in this FIGURE 2 Aroma fingerprint of the control must (unfermented), and the musts partially fermented to 4% (v/v) or 8% (v/v) of ethanol and to which oak chips subsequently were added (1 g/l or 2 g/l) for 30 days. FIGURE 3 Principal component analysis carried out on the OAVs of the odorant terms of the control must (unfermented, C), and the musts partially fermented to 4% (v/v) or 8% (v/v) of ethanol and to which oak chips subsequently were added (1 g/l or 2 g/l) for 30 days.

215 Innovative Elaboration of Sweet Wines component, the tropical fruit, tree fruit, pungent and floral terms showed the highest OAVs, distinguishing them even more clearly from the unfermented musts. Thus, the musts partially fermented to 8% (v/v) ethanol displayed a more intense aroma than the unfermented musts, which were reminiscent of the aroma of commercial sweet wines from raisins. PC2 explained 15% of the variance and encompassed the terms caramelised and toasted. It discriminated between the musts according to whether they were subjected to accelerated ageing, with those fermented to 4% (v/v) ethanol exhibiting the highest scores. Sensory analysis To evaluate the different conditions of the winemaking process used in sensorial terms, the sweet wines obtained were subjected to sensory analysis. Using the opinion of the judges it was possible to estimate the acceptability of sweet wines for the consumer and compare the products with other typical wines from the Montilla-Moriles region. Fig. 4 shows the primary differences established by the sensory analysis of the partially fermented musts, namely differences in acidity, sweetness, balance and global impression. The musts fermented to 4% (v/v) ethanol were judged sweeter and less acidic than the others. The tasters distinguished between the musts fermented to identical ethanol content but treated with different concentrations of oak chips; thus, the musts fermented in the presence of a 2 g/l concentration were better scored for attributes such as woody and astringent. These results are somehow related to the balance and global impression scores, which were higher for the musts fermented to 8% (v/v) ethanol and aged in the presence of a 2 g/l concentration of oak chips, probably as a result of their reduced sweetness/acidity ratio. This, in combination with the perception of woody and astringent notes, led to higher global impression scores for these sweet wines. FIGURE 4 Spider web graph for the musts partially fermented to a) 4% (v/v) or b) 8% (v/v) of ethanol and to which oak chips subsequently were added (1 g/l or 2 g/l) for 30 days and without chips (WC).

Innovative Elaboration of Sweet Wines 216 CONCLUSIONS The oenological parameters (Table 2) demonstrate that the partial fermentation of musts has developed normally. Furthermore, these parameters differed markedly between unfermented and partially fermented musts. The results reveal that fermentation reduced brownness, while ageing increased it. In this study, the OAVs of the compounds were grouped into nine odorant terms to compare the aroma profile of wines; this provided a simple and practical method by reducing the number of variables representing the aroma fingerprint of the samples. The tropical fruit term had higher OAVs in the musts fermented to 8% (v/v) ethanol; also, the presence of wood chips in the medium introduced the toasted term. The caramelised term showed the highest OAVs in the musts fermented to 4% (v/v) ethanol. 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